A variety of materials, systems and organisms have been the subject of genetic engineering to introduce systems to manipulate DNA.
Abremski et al., Cell, 32: 1301-1311 (1983) disclose a site-specific recombination system of bacteriophage P1. The system consists of a recombination site designated loxP and a recombinase designated Cre. Recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs in the presence of Cre.
Sauer, Molecular and Cellular Biology, 7: 2087-2096 (1987) discloses that the loxP-cre recombination system functions in the yeast Saccharomyces cerevisiae. This system was used to excise a gene located between two lox sites which had been introduced into the yeast genome. Cre was expressed from an inducible yeast GALl promoter and this cre gene was located on an autonomously replicating yeast vector.
Sauer and Henderson, Proc. Natl. Acad. Sci. USA, 85: 5166-5170 (1988) disclose that the loxP-cre recombination system functions in a transient manner in mouse cells in tissue culture. Cre was expressed from an inducible mouse metallothionein promoter, the cre gene being located on a papilloma virus replicon-containing vector. Excision of a gene located between two lox sites on a plasmid that was transiently introduced into cells, or of an insert in a gene of a herpesvirus vector, was demonstrated.
Sauer and Henderson, Nucl. Acids Res., 17: 147-161 (1989) disclose that the loxP-cre recombination system functions in a stably transformed mouse tissue culture cell line. Cre, expressed from a rous sarcoma virus promoter, caused excision of a gene located between two lox sites that were integrated in the mouse cell genome.
Gatz and Quail, Proc. Natl. Acad. Sci. USA, 85: 1394-1397 (1988) disclose that expression of the bacterial tet repressor protein in plant protoplasts in culture in a transient manner results in regulation of a CaMV 35S promoter that has tet operator sequences added to it and is also transiently present in the protoplasts.
Baker et al., Proc. Natl. Acad. Sci. USA, 83: 4844-4848 (1986) disclose that the controlling element called "activator" that is derived from maize can excise itself after being introduced into the tobacco genome. Lassnet et al., Mol. Gen. Genet., 218: 25-32 (1989) disclose that the "activator" element can be separated into two functional components: i) an element with a large internal deletion that cannot excise itself, but can be excised by ii) an element with a terminal deletion that cannot excise. These two components were separately transformed into tomato plants, brought together by genetic crosses, and shown to result in excision of the first component in some cells. This experiment indicates that elements from one plant genome can lead to recombination in heterologous plant cells, however the DNA sequences required for activity of the recombination site are not defined.
It is an object of the present invention to manipulate exogenous DNA once it is resident in the plant cell to enhance the ability to control trait expression in engineered plants. A feature of the present invention is the versatility of the method disclosed herein for producing site-specific recombination of DNA in plant cells in that the method is useful toward a wide variety of applications. These and other objects, features and advantages will become apparent upon having reference to the description of the invention herein.